An analyzing system includes a detection area with a transparent window past which an analyte moves and emits or transmits light. One or more polarizing elements receive and polarize the light into respective two or more different polarization components. One or more optical detectors receive the respective two or more polarization components and generate respective at least two signals in response. A processor is coupled to the optical detectors and configured to determine a polarization status of the light from the analyte based on the at least two signals.
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14. A method comprising:
passing an analyte past a detection area;
receiving light from the analyte at one or more polarizing elements that polarize the light into respective two or more different polarization angle components;
directing the two or more different polarization angle components into one or more optical detectors to generate two or more signals corresponding to the two or more different polarization angle components; and
determining a polarization status of the analyte based on the two or more signals.
1. An analyzing system, comprising:
a detection area comprising a transparent window past which an analyte moves and emits or transmits light;
one or more polarizing elements that receive and polarize the light into respective two or more different polarization components;
one or more optical detectors that receive the respective two or more polarization components and generate respective at least two signals in response; and
a processor coupled to the optical detectors and configured to determine a polarization status of the light from the analyte based on the at least two signals.
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This application is a continuation of U.S. application Ser. No. 17/108,314 filed on Dec. 1, 2020, which is incorporated herein by reference in its entirety.
The present disclosure relates to an apparatus and method for fluorescence polarization detection. In one embodiment, an analyzing system includes a detection area with a transparent window past which an analyte moves and emits or transmits light. One or more polarizing elements receive and polarize the light into respective two or more different polarization components. One or more optical detectors receive the respective two or more polarization components and generate respective at least two signals in response. A processor is coupled to the optical detectors and configured to determine a polarization status of the light from the analyte based on the at least two signals.
In another embodiment, method involves passing an analyte past a detection area and receiving light from the analyte at one or more polarizing elements that polarize the light into respective two or more different polarization angle components. The two or more different polarization angle components are directed into one or more optical detectors to generate two or more signals corresponding to the two or more different polarization angle components. A polarization status of the analyte is determined based on the two or more signals.
In another embodiment, a method involves moving a cell marked with a fluorescent dye through a transparent channel and illuminating the cell with linearly polarized light causing light to luminesce from the biological sample. The light luminescing from the biological sample is separated into two or more different polarization angle components. The two or more different polarization angle components are directed into one or more optical detectors to generate two or more corresponding signals. A polarization of the cell is determined based on the two or more signals the cell is classified based on the polarization and a luminescent intensity of the cell.
These and other features and aspects of various embodiments may be understood in view of the following detailed discussion and accompanying drawings.
The discussion below makes reference to the following figures, wherein the same reference number may be used to identify the similar/same component in multiple figures.
The present disclosure is generally related to analyzing sample (e.g., biological samples) using measurements of light that is emitted and/or transmitted from the sample. One example of this type of method is cytometry, which is the measurement of cell characteristics. For example, quantifying the level of fluorescence polarization (Fpol) of particles (e.g., cells) in a flow cytometer can provide information on multiple aspects of the particle, including membrane fluidity, cytoplasmic microviscosity, binding affinities/activities of molecules of interest inside the particle, etc. This information can be further utilized for cancer detection, cell subpopulation identification, and cell activation response monitoring.
Embodiments described below include an analyzing system that is capable of high-sensitivity, high-speed (>10-50 kHz) polarization detection. This system may be used to measure optical characteristics of any type of analyte (e.g., cells, biological samples, living organism, bacteria, beads, droplets). In some embodiments, it may be used for flow cytometry. For example, two applications under consideration for using the analyzing system include using the fluorescence polarization (Fpol) of Methylene Blue to perform cancer detection/rare CTC detection and using the Fpol of DNA staining dye to correct for staining condition variation in sperm sorting.
Fluorescence polarization measurement in a flow cytometer has been used for decades. The molecular orientation and mobility and the energy transfer between fluorophores are major factors that tune the Fpol. For DNA staining dyes, researchers found Fpol revealed the staining condition. Some diseases were shown to have a fluorescence polarization sensitivity due to change of cellular structure and membrane fluidicity.
The effort in exploring fluorescence polarization in flow cytometer seems to be stagnant since 2000. This may be due to rapid development in fluorophore-conjugated biomarkers that make intensity measurement in multiple color channels dominate the flow cytometry apparatus and experiment design. Another development that has reduced interest in exploring fluorescence polarization is rapid development in polarization imaging technologies that reveals polarization information with high spatial resolution. Since Fpol is affected by multiple factors, multimodal information is always preferred. But detectors suitable for the flow cytometer application are usually expensive and bulky, which prevent the easy demonstration of multi-modal flow cytometric detection.
Being able to capture the polarization information in a flow cytometer setting is still of great interest. On one hand, flow cytometry offers easy access to large number statistics and sorting. On the other hand, Fpol offers supplementary information on the particles under investigation, which will provide insight into analysis and sorting for flow cytometry applications. As described below, an analyzing system includes features that facilitate measuring polarization status for purposes such as flow cytometry, and may be useful for other types of analyses where polarization statistics of analytes is useful.
In
The detection area 106 may be a channel that is transparent to the wavelength of the light 108, or has a transparent window on at least one side. The analyte 104 may give off its own light (e.g., bioluminescence, chemiluminescence, with intrinsic anisotropy of emission dipole). In other embodiments, the analyte 104 may be photoluminescent and externally illuminated by a light source (see
Photomultiplier tubes (PMTs) are often adopted as optical detectors in flow cytometers. For example, BD Influx™ system offers a polarization-sensitive detection module based on two PMTs mounted under Brewster angles. Some embodiments described below use silicon photomultipliers (SiPMs) instead of PMTs for polarization sensitive detection for flow cytometry optical detection. Compared to PMTs, SiPM has some advantages in forming a polarization detector. For example, SiPMs operate under relative low voltage (˜30V vs >100V for PMTs). Also, SiPMs are insensitive to magnetic fields, are mechanically robust, have uniform response across the entire sensing area and little variance between different detectors. Generally, SiPMs are compact in size and are easy to build into arrays for compact multi-parameter detection modules. SiPMs are also less costly per detector channel by a factor of 5-10× compared to PMTs. While the embodiments below are described using SiPM detectors, in some embodiments, a semiconductor array using photodiodes and avalanche photodiodes may also be used in place of SiPM.
Use of a SiPM array allows for improved Fpol sensitivity because of their small size/same detection path/closely spaced detectors/insensitivity to misalignment. A SiPM-based fluorescence-polarization detection module can be combined with position correction. This allows for improved fluorescence-polarization detection, e.g., for rare cancer detection where typically a larger sample volume has to be measured. By using a 2×2 (or larger) SiPM array, two or more detector elements can be used to determine the Fpol.
In some embodiments, Fpol detection can be combined with a spatial modulation technique to gain image information (e.g., size and shape) and color co-localization. Some embodiments can be used to design a compact all-in-one detector suitable for Fpol analyzing system. This analyzing system may be designed to minimize the loss in polarization decomposition, e.g., by using polarization beam splitters or birefringence prisms, thus preserving as many as possible photons for detection. The analyzing system can be applied in a wider intensity range as a result. Dim fluorescence signal can also be measured in a polarization-sensitive manner such as in auto-fluorescence cases. A small footprint allows the detector to fit into tight space and be readily adapted to many commercial flow cytometers.
In
A sample/analyte 204 passes through the channel 203 as indicated by arrow 206. The light 202 causes fluorescent emission of the analyte 204 (e.g., optically excites a dye contained within the analyte). This causes emitted light 208 to leave the channel, which includes the fluorescent emissions and may also include some amount of the excitation light 202. The stray excitation light 202 may be filtered by a fluorescence bandpass color filter 210, which selects a fluorescence band of interest for polarization measurements.
The filtered emitted light 211 is input to a polarization beam splitter 212, which directs part of the light to a mirror 214. The light components exiting the beam splitter 212 and mirror 214 may be passed through additional polarizers 216, 218 which further reject light leaking from unwanted directions. Two polarized components 220, 221 of the light illuminate respective optical detectors, which are here shown as SiPM arrays 222, 223 for purposes of illustration. The SiPM arrays 222, 223 produce electrical signals 224, 225 that are received by a processor 226. The processor 226 determines a polarization status of the emitted light 208, which may reflect, for example, a fluorescence polarization of the analyte 204.
The two SiPM detector arrays 222, 223 shown in
Generally, the system 200 decomposes the incoming light according to its polarization components and directs each polarization component 220, 221 onto one SiPM 222, 223 for detection. Both detectors 222, 223 may have individual amplification circuits (e.g., transimpedance amplifiers) and individual digitizer channels. For some embodiments, one polarization detection direction is tuned parallel to the excitation polarization direction of the source light 202 and the other perpendicular to this polarization direction. Equation (1) is used to determine the polarization of the incoming light, where I∥ and I⊥ respectively denote the light intensity measured in the channel parallel and perpendicular to the excitation polarization direction:
As shown in
Generally, the system may provide means to perform alignment and calibration of the detector prior to real test. The two SiPMs 222, 223 should give identical response to unpolarized light. For example, when a fixed intensity, linearly polarized calibration light is shone onto the detector, two SiPMs 222, 223 should provide the same output voltage when the calibration polarization direction is tuned parallel to each detector's detection direction. Imbalanced output can result from un-matched loss in each optical path. A bias-controlled voltage of each SiPM 222, 223 can be adjusted accordingly to compensate. The calibration procedure can take advantage of the SiPM's homogeneous sensitivity across sensor area and linear dependency of gain on control voltage. This may be used in applications where the measured polarization value is to be compared across different instruments/laboratories. For Fpol detection with high resolution, the fluorescence decomposition should be done as close to the detector as possible. The small size of the SiPM arrays enables various approaches for fluorescence decomposition close to the individual detector elements.
Note that while the systems shown in
One example using the four polarization measurements is shown in the diagrams of
In
The power signal 505 has four different levels P1, P2, P3, P4 corresponding to different component through polarizers/wave plates 500-503. In this case the elements of the Stokes vector are given by Equations (2.1)-(2.4) below
S0=P3+P1 (2.1)
S1=P3−P1 (2.2)
S2=2P2−S0 (2.3)
S3=S0−2P4 (2.4)
Many other arrangements of polarizers/wave plates are possible with the constraints that (1) a quarter wave plate is used, (2) reorientation of either the polarizer, quarter wave plate, or both between measurements, and (3) no fewer than four measurements are made. Other arrangements may have better performance with regards to noise or tolerance to misalignments of the rotational axes of the polarizers/quarter wave plates. In
In
In this example, color filters 708-711 are shown over respective optical detectors 700-703 and polarizers 704-707. Further, filters 708-709 affect a first color spectrum and filters 710-711 affect a second color spectrum. In this way, the arrangement in
One of the advantages of characterizing fluorescence polarization using S is that it distinguishes circularly polarized light from unpolarized light. This contrasts with the common method using the “polarization” P=(P90−P0)/(P90+P0), where P90 and P0 are the powers measured with a linear analyzer at 90° and 0°, which implicitly lumps together the unpolarized components and circularly polarized components. The common method works because fluorescence emission is mainly composed of linearly polarized and unpolarized light. However, several mechanisms can give rise to a circularly polarized component in the fluorescence emission including birefringence, circular dichroism, and circularly polarized luminescence. Indeed, circularly polarized luminescence is sometimes defined to be equal to Pl−Pr, the difference between the power with a right/left hand circular analyzer, which is the same as S3.
Determining the circularly polarized component may be valuable either because (1) it contains some valuable information about the cell population, or (2) it contains some information about a noise source. For example, sperm cells are known to be birefringent with the optic axis along the long axis of the head. For sperm sorting, variations in the orientations of the sperm in the interrogation region are a noise source. Using an excitation with a polarization aligned with the fluid stream, the polarized component of the fluorescence is also linearly polarized for an oriented sperm as shown in
Regarding information about the cell population, some reports indicate that sperm birefringence correlates with viability. Thus a measurement of the birefringence has a high value because it can indicate viability. The birefringence may be measured by using an excitation that is polarized at 45° to the vertical. For a sperm with low/no birefringence, the polarized part of the fluorescence is linearly polarized at 45°, as shown in
To calculate the magnitude of this effect, the Jones matrix M for a birefringent sample with a retardance η and with eigen-axes oriented at 45° with respect to the emission polarization is given by Equation (3) below.
In this coordinate system the excitation polarization is (1, 0), so after the sample, the polarization is (1, −iη/2). Decomposing this into the circularly polarized components, it can be shown that the Il−Ir=η. A sperm with a birefringent retardance of 1 nm at an emission wavelength of 480 nm will thus exhibit a fluorescence difference of around 0.3% between the l and r circular channels, assuming the overall output is 50% polarized and 50% un-polarized. This small difference in fluorescence would be difficult to measure. Birefringence has been reported for a variety of cell types including red blood cells, sperm cells and neurons. Red blood cells have much higher birefringence than sperm cells, up to 10 nm has been recorded. It has been shown that the retardance can change by as much as two times as a result of a malaria infection.
The embodiments described herein can be applied in cancer research and cancer detection. Methylene blue (MB) was reported to be a quantitative marker for breast and brain cancer detection using fluorescence polarization imaging at single cell level. However, imaging can only be applied in limited field of views thus limiting number of cells. And there is currently no compatible technology for high speed sorting based on imaging results, preventing large-scale downstream analysis. It is promising to adapt this FDA-approved dye in flow cytometry measurement. Methylene blue stained cells (from liquid biopsy sample or from controlled experiment) are run through a flow cytometer integrated with the proposed detection module. Fluorescence polarization of each cell can be quantified according to Equation (1). Based on the quantitative results, cells can be sub-grouped accordingly or control variables can be investigated statistically. Drug screening and rare population identification/isolation will be greatly benefit.
The embodiments described herein can be applied in XY sperm sorting application where DNA binding dyes (e.g., Hoechst 33342) are used to display the difference in DNA amount between sperms containing X/Y chromosome. Uncertainty exists in the staining process that will slightly change the ratio between the number of DNA base pairs and the number of dye molecules actually bound from cell to cell. As a result, noise will be added onto the ideally linear relationship between the DNA amount and measured fluorescence intensity. The noise will result in broadening of the X/Y population distribution and poorer peak-valley-ratio (PVR) which is a parameter indicative of good quality sorting to happen. Different DNA/dye ratio will result in different inter-dye distance, which will cause different level of energy transfer between dyes. The smaller the DNA/dye ratio, the smaller the effective distance between dyes, the stronger the energy transfer will be. Energy transfer will depolarize the fluorescence signal from DNA binding dyes. Using the proposed polarization detector, fluorescence intensity can be derived by I=I∥+I⊥ while fluorescence polarization P is measured simultaneously. As depicted in
In
To correct for that, the binding ratio, α, can be quantified by the measured fluorescence polarization, P, as shown in graph 1100. It is most true in cases where binding sites are very close to each other (within 10 nm), e.g., DNA binding dyes. When the staining level is high, the average distance between two dyes can fall below 10 nm, in which case, fluorescence resonance energy transfer (FRET) can happen between the two dyes, which will depolarize the fluorescence emission. Thus, by adjusting the measured fluorescence intensity according to P, a better indication can be obtained of the true count of the binding sites in these two cells.
Generally, this involves measure fluorescence polarization, e.g., as indicated by example measurement 1104. Referring to the polarization/staining level characterization curve in graph 1100, the staining level can be determined. Note that the data in graph 1100 can be pre-characterized or estimated using the samples. Using the staining level (0.5 in this example) the corresponding fluorescence correction coefficient 1106 is checked at graph 1102 for given staining level. The measured fluorescence is then corrected using the correction coefficient 1106. Graph 1102 indicates that measured fluorescence intensity is linearly proportional to the staining level given a fixed amount of binding sites.
In another case, there can be free dyes present in the volume under interrogation which affect the measured fluorescence intensity. Under linearly polarized excitation, free dyes will have more depolarized fluorescence emission compared to the bound dyes. By measuring the fluorescence polarization, the free/bound dye ratio can be determined and the fluorescence intensity from the bound dyes can be corrected, which represent the number of binding sites present in the cell. An example of this is shown in
In
In
In
The various embodiments described above may be implemented using circuitry, firmware, and/or software modules that interact to provide particular results. One of skill in the arts can readily implement such described functionality, either at a modular level or as a whole, using knowledge generally known in the art. For example, the flowcharts and control diagrams illustrated herein may be used to create computer-readable instructions/code for execution by a processor. Such instructions may be stored on a non-transitory computer-readable medium and transferred to the processor for execution as is known in the art. The structures and procedures shown above are only a representative example of embodiments that can be used to provide the functions described hereinabove.
The foregoing description of the example embodiments has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the embodiments to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. Any or all features of the disclosed embodiments can be applied individually or in any combination are not meant to be limiting, but purely illustrative. It is intended that the scope of the invention be limited not with this detailed description, but rather determined by the claims appended hereto.
Kiesel, Peter, Johnson, Noble M., Chamoun, Jacob N., Chen, Qiushu, Shi, Norman Nan
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